High-performance liquid chromatographic determination of vitamin D3 in fish liver oils and eel body oils

Identification and determination of vitamin D3 (or D2) and 25-OH-D3 in fish liver oils and eel body oils were carried out. By co-chromatography on HPLC, UV spectra and/or GC-MS, vitamin D3 was identified in naturally occurring fish liver oils and eel body oils, whereas a drop of fish liver oil contained supplemented vitamin D2. 25-OH-D3 was identified only in skipjack liver oil. The HPLC method proposed in a previous report (Takeuchi, A. et al. (1984): J. Nutr. Sci. Vitaminol., 30, 11-25) was confirmed to also be useful for determination of vitamin D3 (or D2) in fish liver oils and eel body oils. The assayed values of vitamin D3 in skipjack and tuna liver oils were 57,760 and 16,200 IU/g, respectively, which were much higher than those in cod and pollack liver oils. The assayed values of vitamin D3 in eel body oils were very low (16-43 IU/g) and showed no appreciable change despite differences in the farming conditions. Determination of 25-OH-D3 in skipjack oil was performed by using HPLC, and the assayed value was 1.8 micrograms/g. This was about 1/800 lower than that of vitamin D3.     

Gas-liquid chromatographic determination of vitamin D in multivitamin preparation.

Gas-liquid chromatographic (GLC) determination of vitamin D in multivitamin preparations was investigated and a simple routine method was established. The unsaponifiable matters of a sample was applied to a thin-layer chromatography (TLC) plate using Kieselgel GF254 as an adsorbent and a mixture of n-hexane-ethyl acetate (4:1) as a developing solvent. The scraped zones corresponding to vitamin D and previtamin D were extracted and determined by GLC using 1.5% OV-17 packed on Shimalite W (80-100 mesh) as a stationary phase. When the proposed method was applied to model preparations made by mixing vitamin D, A and E, it was confirmed that the determination of vitamin D was possible when the ratios of vitamin A and E (DL-alpha-tocopheryl acetate) to vitamin D were within 104 (I.U. ratio) and 2,500 (weight ratio), respectively. Since the ratios of most of commercial multivitamin preparations on sale in Japan are within the limitations and the results on multivitamin preparations were also satisfactory, the proposed method was confirmed to be suitable simple routine.     

Gas-liquid chromatographic determination of vitamin D in multivitamin preparations containing excess amounts of vitamin E.

Gas-liquid chromatographic (GLC) determination of vitamin D in multivitamin preparations containing excess amounts of vitamin E (a more than 2,500 weight ratio of dl-alpha-tocopheryl acetate to vitamin D) was investigated and a simplified routine method was established because the method reported previously (1) could not be applied to such special preparations. After applying the unsaponifiable matters of a sample to a phosphate-treated alumina column chromatography prepared according to MULDER et. al. (2), the eluate was evaporated and the residue was subjected to thin-layer chromatography (TLC) and GLC. When this method was applied to model preparations made by mixing vitamin D2 and dl-alpha-tocopheryl acetate (excess amounts), good results were obtained. Since the results on a commercial multivitamin preparation containing excess amounts of vitamin E were also satisfactory, it was confirmed that the proposed method could be used for simplified routine determinations.     

Naturally occurring vitamin D3 in fish products analysed by HPLC, using vitamin D2 as an international standard.

A chemical method for the analysis of naturally occurring vitamin D is proposed. The unsaponifiable matter of oils and tissues is prepared, cholesterol is partly removed by double precipitation at low temperature in methanol. The vitamin D fraction is collected on an adsorption column by high performance liquid chromatography. The fraction is further purified and the vitamins D2 and D3 are separated on a partition column (reverse phase) by HPLC. Recovery was 89 to 93%, standard deviation 3%. The only vitamin D analogue found in fish oils, livers and fillets, was cholecalciferol (D3). Hence, ergocalciferol (D2) could be used as an internal standard. The provitamins ergosterol and 7-dehydrocholesterol, as well as the previtamins, were separated from the vitamin D-fraction on the adsorption column. Results in the range 0.050 to 134 microgram D3 per gram (2 to 5360 I.U. per gram) are given. One cod liver oil was analysed in a rat bioassay, giving supporting results.     

25-hydroxyvitamin D3 affects vitamin D status similar to vitamin D3 in pigs--but the meat produced has a lower content of vitamin D

In food databases, the specific contents of vitamin D3 and 25-hydroxyvitamin D3 in food have been implemented in the last 10 years. No consensus has yet been established on the relative activity between the components. Therefore, the objective of the present study was to assess the relative activity of 25-hydroxyvitamin D3 compared to vitamin D3. The design was a parallel study in pigs (n 24), which from an age of 12 weeks until slaughter 11 weeks later were fed approximately 55 microg vitamin D/d, as vitamin D3, in a mixture of vitamin D3 and 25-hydroxyvitamin D3, or 25-hydroxyvitamin D3. The end-points measured were plasma 25-hydroxyvitamin D3, and in the liver and loin the content of vitamin D3 and 25-hydroxyvitamin D3. Vitamin D3 and 25-hydroxyvitamin D3 in the feed did not affect 25-hydroxyvitamin D3 in the plasma, liver or loin differently, while a significant effect was shown on vitamin D3 in the liver and loin (P < 0.001). 25-Hydroxyvitamin D3 in the plasma, liver and loin significantly correlates with the sum of vitamin D3 and 25-hydroxyvitamin D3 in the feed (P < 0.05). Therefore, 25-hydroxyvitamin D3 should be regarded as having the same activity as vitamin D3 in food databases. Sole use of 25-hydroxyvitamin D3 as a vitamin D source in pig feed will produce liver and meat with a negligible content of vitamin D3, while an increased content of vitamin D3 in the feed will produce liver and meat with increased content of both vitamin D3 and 25-hydroxyvitamin D3.     

Determination of vitamin D3 in margarines, oils and other supplemented food products using HPLC

The vitamin D3 content of high fat foods, such as margarines and oils, was determined by liquid chromatography. The method involved sample saponification and an extraction process, in which the purification of the non-saponifiable matter was the critical step. A semi-preparative straight-phase clean up procedure was used to produce an adequately purified sample solution prior to the final analytical quantification step. Vitamin D2 was used as internal standard when estimating the vitamin D3 content. The overall recoveries depended on the fat content of the food sample and varied from 40% for margarines to 80% for low-fat products at a concentration of about 10 micrograms/100 g. The minimum detectable amount was 0.36 ng vitamin D3. The mean relative recovery of vitamin D3 in relation to vitamin D2 was calculated to be 0.97.     

High-performance liquid chromatographic determination of vitamin D in foods, feeds and pharmaceuticals by successive use of reversed-phase and straight-phase columns

A simplified and accurate method for the determination of vitamin D in foods, feeds and pharmaceuticals was established by high-performance liquid chromatography (HPLC) using successively reversed-phase and straight-phase columns. About 1-2 g of a sample was accurately weighed and directly saponified. The extracted unsaponifiable matter was subjected to preparative HPLC using a reversed-phase column and a vitamin D fraction was collected. The fraction was subsequently subjected to analytical HPLC using a straight-phase column and vitamin D was assayed by estimating the peak height. The proposed method was applied to various kinds of samples, e.g., fishery products, fish meals, mixed feeds for fish farming and chicken farming, egg yolk, milk products, cattle liver, Shiitake, fortified foods and multivitamin preparations. The results showed that the proposed method was useful for the determination of vitamin D in such samples, because the peak of vitamin D in the profile of the second HPLC was always clearly separated from other concomitants and good recovery was obtained. Therefore, we think that the method is useful as a routine one for the determination of vitamin D in foods, feeds and pharmaceuticals.     

Simplified assay of vitamin D2 in fortified dried milk by using two steps of high-performance liquid chromatography

A simplified method for the determination of vitamin D2 in fortified dried milk was established by using two steps of high-performance liquid chromatography (HPLC). About 1 g of fortified dried milk was accurately weighed and directly saponified. The extracted unsaponifiable matter was first subjected to preparative HPLC using a Nucleosil 5C18 column (reversed-phase type) with acetonitrile-methanol (1:1) as a mobile phase and a fraction containing vitamin D2 was separated. The separated fraction was subsequently subjected to analytical HPLC using a Zorbax SIL column (straight-phase type) with 0.4% isopropanol in n-hexane as a mobile phase. Since the peak corresponding to vitamin D2 was clearly observed with separation from other concomitants on the chromatogram of the analytical HPLC, the vitamin was assayed by estimating the peak height. The overall recovery of vitamin D2 by the proposed method was 94.3 +/- 2.3% (mean +/- SD). Naturally occurring vitamin D3 derived from cow's milk was negligible in commercial fortified dried milk sold in Japan. When the proposed method was applied to 5 kinds of commercial fortified dried milk, satisfactory results were obtained.     

化妆品中维生素D2、维生素D3的高效液相色谱测定

目的建立化妆品中维生素D2、维生素D3的高效液相色谱检测方法,用于监测化妆品中维生素D2、维生素D3的使用情况.方法通过考察不同型号液相色谱柱、不同类型流动相及配比,确定了最佳色谱条件,并进行了线性范围、检出限、精密度、准确度试验.结果在Alltima C18(250mm×4.6mm I.D.,5μμm)色谱柱;流动相为甲醇乙腈=9010;流速为1.0 ml/min;检测波长为265nm;柱温为25℃时,维生素D2、维生素D3在0.5～100mg/L浓度范围内具有良好的线性关系,检出限分别为0.12mg/L和0.06mg/L;不同浓度维生素D2、维生素D3的变异系数分别小于3.8%和3.5%;加标量为20μg/g时,维生素D2、维生素D3的加标回收率分别在94.2%～101.4%和91.6%～97.2%之间.结论该方法具有操作简便、准确、快速等特点,适于同时测定化妆品中的维生素d2、维生素D3.     

Identification of vitamin D3 and 7-dehydrocholesterol in cow's milk by gas chromatography-mass spectrometry and their quantitation by high-performance liquid chromatography.

Identification of vitamin D3 and 7-dehydrocholesterol (7-DHC) in cow's milk by gas chromatography-mass spectrometry (GC-MS) and their quantitation by high-performance liquid chromatography (HPLC) were investigated. When vitamin D and provitamin D fractions purified from a sample of commercial cow's milk were applied to GC-MS, the results showed that the fractions contained vitamin D3 and 7-DHC, respectively, while neither vitamin D2 nor ergosterol could be detected in the milk. HPLC methods for the determination of vitamin D3 and 7-DHC in cow's milk were then proposed as routine methods. The method for assaying vitamin D3 included the isolation of lipids from milk according to the directions of Bell and Christie (5), saponification, isolation of unsaponifiable matter, digitonin-Celite column chromatography, preparative thin-layer chromatography (TLC) and application to HPLC. On the other hand, 7-DHC in milk could be simultaneously determined without the purification by digitonin-Celite column chromatography. The peak corresponding to either vitamin D3 or 7-DHC in the respective HPL-chromatograms was clearly separated from possible interfering substances and the recovery experiments for both vitamin D3 and 7-DHC gave satisfactory results. When the proposed methods were applied to 10 samples of commercial cow's milk, the assayed values of vitamin D3 and 7-DHC were 19--79 I.U./liter and 14--56 microgram/liter, respectively.     

A rapid and precise method for the determination of vitamin D3 in rat skin by high-performance liquid chromatography.

In order to develop the investigations into photobiogenesis of vitamin D3, a rapid and precise method for the determination of the vitamin in rat skin was established by using high-performance liquid chromatography (HPLC). The proposed method included saponification of small pieces of rat skin, extraction of the unsaponifiable matter and application to HPLC using "Zorbax SIL" (straight-phase) as an adsorbent and 0.5% isopropanol in n-hexane as a mobile phase. The applicable lower limit of the method was 2ng of vitamin D3/cm2 of subcutaneous tissue-removed skin and it was possible to assay a concentration higher than 2 ng/cm2. The proposed method was applied to determine the content of vitamin D3 in rat skin obtained from in vivo and in vitro irradiation experiments. In the in vitro experiment, the yield of vitamin D3 increased in proportion to the irradiation time. On the other hand, the yield in the in vivo experiment showed a proportional increase similar to the in vitro experiment until 60 min irradiation, while a nearly constant value was obtained by irradiation for longer than 60 min. When the rat skin obtained from the in vitro experiment was irradiated with monochromatic UV rays in the range s60-350 nm, the most effective wavelength for the formation of vitamin D3 was confirmed to be 303 nm, which differs from the result obtained from the experiment in a test tube (295 nm). Moreover, the yield of vitamin D3 by irradiation with UV rays below 288 nm was extremely low, which again differed from the results of a test tube experiment. These differences were thought to be due to the filter effect of the malpighian layer in the epidermis of rat skin.     

Occurrence of vitamin D sulfate in human milk whey

Following reports that vitamin D sulfate is the major source of vitamin D activity in human milk, we investigated the presence of this compound in milk whey using a modification of techniques for the determination of vitamin D metabolites in plasma. Synthetic cholecalciferol sulfate, ergocalciferol sulfate an [3H]cholecalciferol sulfate were prepared by reacting radioactive cholecalciferol or nonradioactive cholecalciferol or ergocalciferol with sulfamic acid in pyridine. The products were purified sequentially by Sephadex LH-20 and high pressure liquid chromatography. The purified products were chromatographically homogeneous, exhibited an ultraviolet absorption spectrum identical to that of standard cholecalciferol, demonstrated a sulfonate ester linkage and upon saponification yielded the parent vitamin. Milk whey was extracted with methanol:methylene chloride (1:2 v/v) using [3H]cholecalciferol sulfate to estimate recovery of the compound. The extract was purified by chromatography on silica cartridges an reverse phase high pressure liquid chromatography and was quantitated by ultraviolet absorption (UV). Although added cholecalciferol sulfate was readily detected in human milk whey samples, no endogenous vitamin D sulfate was found (detection limit 1 ng/ml). The results indicate that vitamin D sulfate is not a major source of vitamin D activity in human milk.     

High-performance liquid chromatographic determination of vitamin D3 in bovine colostrum, early and later milk

A simplified and accurate method for determination of naturally occurring vitamin D3 in bovine milk was established by high-performance liquid chromatography (HPLC) using successively reversed-phase and straight-phase columns. Exactly 25.0 ml of a sample of bovine milk was taken and the lipid was extracted with a solvent mixture of petroleum ether and ethyl ether (1:1) with small amounts of ethanol and Triton X-100, present. The extracted lipid was subjected to the first preparative HPLC using a Nucleosil 5C18 column (reversed-phase type) with acetonitrile-methanol (1:1) as the mobile phase, and a fraction containing vitamin D3 was isolated. The fraction was subsequently subjected to the second analytical HPLC using a Zorbax SIL column (straight-phase type) with 0.4% isopropanol in n-hexane as the mobile phase. Vitamin D3 was assayed by estimating the peak height on the chromatogram. The overall recovery and CV values were 92.1 +/- 8.7% and 9.4%, respectively, which were satisfactory. The proposed method was applied to several kinds of colostrum, early and later bovine milk in pairs. The assayed values in the colostrum of the group with large amounts of vitamin D3 administered before delivery to prevent parturient paresis were higher than those in the group with no administration. However, the values of the former group generally decreased in the respective early and later milk in step and there were few differences among those in the later milk of the two groups. The assayed values in later milk were 30-80 IU/liter.     

Mammary transfer of vitamin D3 in the rabbit doe

Mammary transfer of vitamin D3 from a single intravenous injection containing 100 000 IU vitamin D3 in ethanol, was studied in lactating rabbit does. Significant amounts of the injected vitamin D3 appeared in the milk of does. The highest concentration was observed in the first milk sampling (24 h after administration) which was followed by a decreasing pattern in the five postinjection days. Vitamin D3 concentration in the neonate tissues were smaller than in their dams. In the maternal tissue the highest concentration occurred in the liver, whereas muscle had the lowest value. Among the various neonatal tissues liver contained the highest amounts although in the other tissues appreciable amounts of vitamin D3 were recovered.     

In vivo and in vitro conversion of 7-dehydrocholesterol into vitamin D3 in rat skin by ultraviolet ray's irradiation.

In order to confirm the photochemical conversion of 7-dehydrocholesterol (7-DHC) into vitamin D3 in rat skin, the following in vitro and in vivo experiments were carried out. In the first (in vitro) experiment, the skin stripped off from a sacrificed normal rat was irradiated with an ultraviolet (UV) lamp for a constant period. In the second (in vivo) experiment, the normal rat, irradiated under the same condition mentioned above, was sacrificed and then the skin was stripped off. Lipids were individually extracted with chloroform-methanol (1:1) from the skin obtained in the two experiments and the solvent was evaporated. The resulting residue was saponified and the unsaponifiable matter extracted with benzene was purified by application to hydroxyalkoxy-propyl (HAP) Sephadex column chromatography. The resulting purified vitamin D3 fraction was applied to high-performance liquid chromatography (HPLC) in order to estimate vitamin D3. No peak, aside from that of alpha-naphthol as an internal standard, was observed in the HPLC chromatogram on the skin obtained from the non-irradiated rat, whereas the peak corresponding to vitamin D3 was observed in each HPLC chromatogram on both the irradiated skin (in vitro experiment) and the skin obtained from the irradiated rat (in vivo experiment). The peaks, confirmed to be due to vitamin D3 by the results of co-chromatography, were increased according to the increase of irradiation energy and there were little differences between the corresponding estimated values of vitamin D3 in the two experiments. These results prompted the conclusion that 7-DHC in rat skin was photochemically converted into vitamin D3 by UV irradiation and that the in vivo conversion mechanism might be the same as the in vitro one.     

Rapid HPLC method for measurement of vitamin D3 and 25(OH)D3 in blood plasma

The present work describes a novel, simplified high-performance liquid chromatography (HPLC) method for evaluation of vitamin D3 and its 25-hydroxy metabolite in blood plasma. The retrieval of the analytes from the blood plasma matrix is based on a single-step extraction using acetonitrile. The method is specific, sensitive, and ensures good reproducibility. The recovery of the analytes, precision, and reproducibility obtained using the present approach gave results comparable to or better than more complex, laborious, and time-consuming procedures. This method is suitable for evaluation of the host's vitamin D physiological status, as well as for rapid analysis of blood plasma samples in suspected cholecalciferol toxicity. With a significantly shortened time of analysis (10 minutes), the present method allows the possibility for processing of a large number of samples rapidly, efficiently, and at a low cost.     

An improved procedure for the isolation of suprasterol2 I and II from a photochemical reaction mixture of ergocalciferol (vitamin D2).

An improved procedure for the isolateion suprasterol2 I and II from a photochemical reaction mixture of ergocalciferol (vitamin D2) and their spectral data are described in this paper. When a solution of ergocalciferol in ethanol was irradiated by UV light from a high-pressure mercury lamp, the reaction mixture gave six spots, including suprasterol2 I and II, on the thin-layer chromatogram, while the peaks corresponding to pyro-D2, isopyro-D2,5,b-trans-D2, suprasterol 2 I and II were observed in the gas chromatogram obtained from a capillary column GLC (Suprasterol2 I and II were main peaks). After purifying the mixture by column chromatography on silica gel containing 12% alumina as an absorbent, two main fractins were isolated. The data of their spectra, TLC and GLC showed that the former fraction was suprasterol2 II while the latter was suprasterol2 I and that the both fractions contained the respective compound only. Both suprasterol2 were crystallized as the 3,5-dinitro-benzoates.     

Binding of 7-dehydrocholesterol to sterol carrier protein and vitamin D3 effect.

It was confirmed that delta 5,7-sterol delta 7-reductase activity was suppressed by cholecalciferol (vitamin D3) in the enzyme system consisted of microsomes and sterol carrier protein (SCP). The enzyme activity was significantly decreased in the combination with microsomes obtained from either vitamin D-deficient or vitamin D3-treated rat liver and with SCP obtained from vitamin D3-treated rat. It was also demonstrated by the binding assay of the dextran-charcoal technique that 7-dehydrocholesterol binding to SCP could be specifically displaced by vitamin D3. The inhibition of cholecalciferol on 7-dehydrocholesterol binding to liver SCP was confirmed to be non-competitive inhibition.     

Tissue distribution of 7-dehydrocholesterol, vitamin D3 and 25-hydroxyvitamin D3 in several species of fishes

A high-performance liquid chromatographic (HPLC) method for simultaneous determination of 7-dehydrocholesterol (7-DHC), vitamin D3 and 25-hydroxyvitamin D3 (25-OH-D3) in tissues of fishes was established, and using this method the tissue distribution of the sterols in lamprey (Entosphenus japonicus), great blue shark (Prionace glauca), skipjack (Katsuwonus pelamis) and albacore (Thunnus alalunga) was investigated. The results are summarized in the following: Although the alimentary canal, gall bladder and roe of lamprey and the alimentary canal of great blue shark contained comparatively high levels of 7-DHC (higher than 2,000 ng/wet tissue g), the other tissues of lamprey and great blue shark and all tissues of skipjack and albacore contained only low levels of 7-DHC (lower than 1,000 ng/g). There was no significant correlation between the levels of 7-DHC and vitamin D3. The contents of 7-DHC in the skin of skipjack and albacore were only 1/1,000 of those in the skin of rats. Although the contents of vitamin D3 in the liver of skipjack and albacore were extremely high (41,240 and 21,000 ng/g, respectively), those in the skin were very low (454 and 257 ng/g, respectively). 25-OH-D3 was detected in the viscera of skipjack, but the levels were not very high (lower than 150 ng/g). These levels were not significantly correlated with those of vitamin D3. The results suggest that large quantities of vitamin D3 in the liver of skipjack and albacore are supplied by other biosynthetic routes or by intake of vitamin D3 rather than by photochemical biosynthesis.